Recombinant Cellulose-binding Domains: Binding Characterization And Their Application In Improvement Of Paper Properties

In this study, we designed several overlapping primers based on the CBD1 cDNA sequence from Volvariella volvacea cbh 1-1 and the CBM3 cDNA sequence from Clostridium thermocellum cipB, and synthesized double CBD gene fragments by PCR procedure. The fragments were digested and ligated with expression plasmid pET-32b then transferred into E. Coil BL21(DE3). SDS-PAGE analysis showed the recombinant proteins CBM3-GS-CBD1, CBM3-celc-CBD1, CBM3-GS-CBM3 and CBD1-celc-CBD1 were expressed successfully. CBD 1-celc-CBD1 was only soluable at 20℃, while other proteins had soluable expression at 30℃.Recombinant proteins were purified by affinity chromatography using Ni-NTA agarose, and the binding capacity of proteins to Avicel, phosphate swollen cellulose(PASC), Whatman No.1 filter paper and pulp were compared.The results were:(1) It was found that the binding capacity of CBD1 to crystalline cellulose was greatest, and the binding capacity of CBD 1-celc-CBD1 to Avicel was as three times as CBM3-GS-CBM3. The maximal absorption of PASC was CBM3-GS-CBD1,while the maximal absorption of Whatman No.1 filter paper was CBM3-celc-CBDl respectively.And the CBD1-celc-CBDl had the highest binding capacity to paper plup. (2) Linkers had some influence on binding capacity. CBM3-GS-CBD1 containing flexible linker preferred to Avicel whereas CBM3-celc-CBD1 conjuncting by native linker preferred to Whatman No.1 filter paper. (3) In addition, we found CBD 1-celc-CBD1 bonding to Avicel is reversible while the others were irreversibility.We also studied the capability of proteins to improve the treated paper's mechanical properties, found CBM3-GS-CBM3 significantly enhancing treated paper's folding endurance and tensile strength by 27.4% and 15.5% respectively.Similarly CBD1-celc-CBD1 could increase the treated paper's folding endurance by 16.7% and increase it's tensile stength by 12.6%. Interestingly, we found CBM3-GS-CBD1 and CBM3-celc-CBD1 both had improvement on drainability of treated paper. Besides, we also demonstrated that surface hydrophilicity of the proteins treated paper are slightly increased.In this paper, we systematically studied the different binding capacity of CBDs from CBH I-Ⅰand CipB, and discussed their application in paper modifaction for the first time. We also compared the influence of different structure linkers to double CBD fusion proteins. The results obtained showed that recombinant proteins can be used to alter surface and mechanical properties of paper. Besides, the reversibility of CBD1-celc-CBD1's binding to cellulose makes it suitable for fiber recycling. This paper provided the theoretical and practical fourdation for developing a novel paper-modifaction reagent.